Research Paper
Promotive action of lysophosphatidic acid on proliferation of rat embryonic neural stem cells and their differentiation to cholinergic neurons in vitro
CUI Hui-Lin, QIAO Jian-Tian*
Department of Neurobiology, Shanxi Medical University, Taiyuan 030001, China
Abstract: Effects of lysophosphatidic acid (LPA), an extracellular phospholipid signal, on the proliferation of rat embryonic neural stem cells (NSCs) and their differentiation into microtubule-associated protein 2 (MAP2)-positive and choline acetyltransferase (ChAT)-positive, i.e. cholinergic-committed neurons, were observed in vitro by [3H]-thymidine incorporation, immunocytochemistry, Western blot and other techniques. The results showed that: (1) Lower concentrations of LPA (0.01~1.0 μmol/L) dose-dependently enhanced the uptake of [3H]-thymidine by NSCs cultured in specific serum-free medium, indicating a significant promotive action of LPA on the proliferation of NSCs. (2) After fetal bovine serum which induces and commences the differentiation of NSCs, was used in the medium, the lower concentrations of LPA increased the percentages of both MAP2- and ChAT-immunoreactive neurons, with a peak at 0.1 mmol/L LPA in two cases. (3) The promotive effects of LPA on the differentiation of MAP2- and ChAT-positive neurons were also supported by the up-regulation of the expressions of both MAP2 and ChAT proteins detected by Western blot. (4) At the early phase of differentiation of NSCs, the cell migration and neurite extension were enhanced significantly by lower dosages of LPA under phase-contrast microscope. These results suggest that LPA within certain lower range of concentrations promotes the proliferation of NSCs and their differentiation into unspecific MAP2-positive and specific cholinergic-committed neurons, and also strengthens the migration and neurite extension of the newly-generated neuronal (and also glial as reported elsewhere) progenitors.
Key words: differentiation; proliferation; cholinergic neuron; embryonic neural stem cells; lysophosphatidic acid
溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化
崔慧林， 乔健天*
山西医科大学神经生物学研究室，太原 030001
摘 要：溶血磷脂酸(lysophosphatidic acid, LPA)是一种细胞外磷脂信号。本研究用[3H]-胸腺嘧啶掺入法、免疫细胞化学和 Western blot等技术，观察了LPA对体外培养的大鼠胚胎神经干细胞(neural stem cells, NSCs)的增殖以及向MAP2标记的一般神经元和ChAT 标记的胆碱能神经元的分化的影响。结果显示：(1)在特殊的无血清培养基中加入低浓度的LPA (0.01~1.0 μmol/L)后，NSCs对[3H]-胸腺嘧啶的摄入呈剂量依赖性增加，表明LPA对NSCs有显著的促增殖作用；(2)在培养基中加入胎牛血 清以诱导NSCs的分化，发现低浓度的LPA增加MAP2阳性和ChAT阳性神经元的比例， 0.1 mmol/L LPA 引起的增加达到峰值；(3) Western blot分析显示LPA促进了MAP2和ChAT的表达；(4)在诱导NSCs出现分化早期，用倒置显微镜观察到低浓 度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明，低浓度LPA在一定范围内可以促进NSCs的增殖、并分 化为一般的MAP2阳性神经元和特殊的胆碱能神经元，而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞 的迁移和突起生长。
关键词：分化；增殖；胆碱能神经元；胚胎神经干细胞；溶血磷脂酸
中图分类号：Q254；Q189
Neural stem cells (NSCs) are capable of self-renewal and can give rise to neuronal and glial lineages[1-3]. Regulation of the proliferation and differentiation of NSCs in vitro may be important for the potential development of transplantation strategies and other therapeutic approaches in the treatment of neural injuries and neuro-degenerated diseases[4-9]. Studies on the NSC-based neurogenesis and gliogenesis in vitro have suggested that these processes occur by stepwise restriction and are dependent on environmental signals[10-12]. Lysophosphatidic acid (LPA), a simple phospholipid, mainly observed in epithelial cells, exhibits properties as an extracellular growth factor mediating diverse cellular responses including cell proliferation, through transmembrane and intracellular signaling pathways[13,14]. In the nervous system, many investigators have reported that LPA causes the collapse of neuron growth cone and tends to inhibit or reverse the morphological differentiation of many cell lines[15-17], while there is few report on the effects of LPA on the proliferation and differentiation of NSCs[18,19]. For the functional importance of cholinergic neurons in the central nervous system, such as taking part in locomotive, wake/sleep and learning/memory behaviours[20,21], the aim of the present study was designed to clarify the effects of LPA on the proliferation of embryonic NSCs, and especially the differentiation of cholinergic neurons from NSCs of rats in vitro by using [3H]-thymidine incorporation, single or double immunocytochemical labeling combined with Hoechst 33342 staining, Western blot and phase-contrast microscopy techniques.
1 MATERIALS AND METHODS
1.1 Primary culture of embryonic NSCs and neurosphere passaging
E14 rat embryos were obtained from timed-pregnant Wistar rats (from Animal Center of Shanxi Medical University) with the morning of vaginal plug designated as embryonic day 0 (E0). Cultures were produced essentially as described previously[22]. Briefly, the cortex was dissected out and dissociated mechanically with a fire-polished pipette in the proliferation growth medium composed of a 1:1 mixture of DMEM and F12 nutrient (Life Technologies, MD, USA), 20 ng/ml bFGF (Pepro Tech., NY, USA), 10 ng/ml EGF (Pepro Tech.), 0.025 mg/ml insulin (Sigma, USA), and 1×B27 supplement (Gibco-BRL, USA). The dispersed cells were plated at 1×104 cells/cm2. All cells were incubated at 37 oC in 95% air and 5% CO2. Under these conditions, cells grew rapidly to form neurospheres. After 4 d in vitro (DIV), the floating clusters of neurospheres were harvested by gentle centrifugation, mechanically dissociated, and re-plated under the same conditions, until secondary neurospheres were generated. Neurospheres were passaged at least twice to eliminate short-term dividing precursors and bulk cultures were generated prior to use in further studies. Cell counting and viability tests were performed at every passage by Trypan blue exclusion.
1.2 Identification of NSCs by immunocytochemistry
For identification of NSCs, neurospheres were plated in glass coverslips pre-coated with poly-L-lysine (Sigma), grown for 1 d in proliferation growth medium. Then they were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Nestin, a class VI intermediate filament protein, was used as marker protein for identifying NSCs[1,10,23,24]. Mouse anti-nestin (1:1 000, mouse monoclonal, BD Biosciences, USA) was applied overnight at 4 oC, biotinylated secondary IgG (Bioster, China) applied for 1 h, and streptavidin-Cy3 (Bioster) applied for 30 min. For the purity analysis of NSCs, dissociated cells were then incubated further with Hoechst 33342 (Sigma) to visualize the nuclei of all cells other than NSCs only, and thus the purity of NSCs could be calculated.
1.3 DNA synthesis with [3H]-thymidine incorporation
Proliferation of NSCs was assessed by DNA synthesis with [3H]-thymidine incorporation as described previously[25]. Briefly, dissociated cells were grown quiescently in DMEM/F12 for 24 h, then switched to the growth medium containing different concentrations of LPA (Sigma); and 24 h later, cells were exposed to [3H]- thymidine (1 μCi/ml, Shanghai Institute of Applied Physics, China) for 4 h and then the trichloroacetic acid-precipitable material was quantified by liquid scintillation counting. LPA was dissolved in PBS containing 4% fatty acid-free bovine serum albumin (FAFBSA, Sigma) and stored as stock solution (1 mmol/L) at -20 oC.
1.4 Differentiation of NSCs induced by fetal bovine serum (FBS)
To study the differentiation of neuronal progenitors from NSCs, the third passage of neurospheres were transferred onto 35 mm diameter dishes with poly-L-lysine-coated glass coverslips, about 20 neurospheres per dish, and cultured in differentiation growth medium composed of DMEM/F12 and 10% FBS (Gibco-BRL) with different concentrations of LPA or vehicle only at the same time. The FBS-induced differentiation was observed in the following days by immunocytochemical, morphological and Western blot techniques.
1.5 Identification of microtubule-associated protein 2 (MAP2)- or choline acetyltransferase (ChAT)-positive neurons by immunocytochemistry
The double-labeling immunocytochemical procedures for detecting MAP2, a neuronal-specific dendritic marker[26] and ChAT, a marker of cholinergic neurons[27] were performed as described previously[28] with a few modifications. In brief, cells on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min, then washed with PBS before permeabilization in 0.4% Triton X-100 for 30 min. After washed in PBS, cells were blocked in PBS containing 20% goat serum for 60 min. For double-labeling experiments, primary antibodies (mouse monoclonal anti-MAP2 (1:1 000, Sigma) and rabbit polyclonal anti-ChAT (1:400, Chemicon, USA) were diluted in PBS containing 10% normal goat serum and 0.3% Triton X-100. Coverslips were incubated for 2 h at 37 oC and then washed three times with PBS as above. Biotinylated-conjugated secondary antibodies to mouse were added. Cells were incubated for 30 min at 37 oC. After washed for 5 min with PBS three times, coverslips were incubated with streptavidin-Cy3 and FITC-conjugated goat anti-rabbit (Bioster) for 2 h at 37 oC. The coverslips were washed three times again. Finally, for unspecific staining of nuclei of all cells in the specimen, Hoechst 33342 (1.0 mg/ml) was added for 15 min at room temperature, followed by rinse for 5 min with PBS twice. A rapid water washing preceded the mounting on glass slides with glycerol. Then, different fluorescences were detected with an Olympus BX40 microscope and photographed with CoolSNAP digital camera. The photographs were determined with image analysis software IPP 4.5.
1.6 Determination of MAP2 and ChAT proteins by Western blot analysis
The cells in dishes were washed three times in PBS, lysed in 250 μl lysis buffer (50 mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, 0.02% sodium vandate, 0.1% SDS, 0.5% deoxycholic acid, 100 μg/ml PMSF, 0.2 μg/ml leupeptin, 1% NP-40) per dish. Samples were separated by SDS-PAGE on 8.5% (for MAP2) and 8% polyacrylamide gel (for ChAT) respectively, and then transferred onto nitrocellulose filters by a semi-dry blotting system[29]. Unspecific binding was blocked with 6% casein, 1% polyvinyl-pyrrolidone, 10 mmol/L EDTA, and 3 mmol/L NaN3 in PBS for 1 h[30]. After incubation with mouse anti-MAP2 and rabbit anti-ChAT overnight at 4℃, alkaline phosphatase (AP)-conjugated secondary IgG was added for 2 h at room temperature. The blots were detected with AP substrate as described previously[31]. Reagents of Western blot were from Biobasic Inc., Canada.
1.7 Statistical analysis
Each experiment was repeated at least three times. Data were expressed as percentages of the control values (means±SEM). For calculating the percentage changes of two kinds of differentiated neurons, the blue-stained nuclei and red- or green-stained neurons were counted respectively from 20 fields in each experiment, with the number of blue-stained nuclei (i.e., the total cell number to be counted) not less than 200. Data were submitted to a one-way ANOVA followed by Dunnet's post hoc test. Differences were considered statistically significant atP<0.05.
2 RESULTS
2.1 Identification of NSCs and the effects of LPA on proliferation of NSCs
As stated above, 24 h after the dissociated cortices of E14 rat embryos were cultured in the growth medium that prevented differentiation of NSCs, small-sized neurospheres composed of several cells appeared with many dead cells and cell debris. At 3~4 DIV, the neurospheres became more and larger, composed of more cells. After at least two passages (at 15~20 DIV), unattached and dead cells appeared but phase-bright neurospheres suspended in the growth medium (Fig.1). By immunocytochemical examination at 20 DIV, almost all the cells that composed neurospheres exhibited strongly nestin-positive (red-stained), and they were identified as real NSCs. The purity of nestin-positive cells at 20 DIV was more than 96% and they were used for further observation.
By DNA synthesis with [3H]-thymidine incorporation techniques, it was shown that application of LPA exhibited a dual or bi-directional effects on the proliferation of NSCs according to the concentrations of LPA that were used in different groups (Fig.2). Lower concentrations of LPA (0.01~1.0 μmol/L) dose-dependently enhanced the uptake of [3H]-thymidine by NSCs, with the peak response at the concentration of 1.0 μmol/L, showing an increasing count per minute (CPM) of [3H]-thymidine incorporation up to (1.51±0.06) folds compared with that in the vehicle control group (P<0.01). When higher concentrations (>1.0 μmol/L) of LPA were used, the enhancement of [3H]-thymidine uptake declined, and moreover, as the concentrations reached or surpassed 2.0 μmol/L, application of LPA even caused decrease of NSCs as compared to that in the control group.
2.2 Effects of LPA on neuronal differentiation of NSCs
2.2.1 Detection of MAP2- and ChAT-positive neurons
The third passage of NSCs was transferred into differentiation growth medium containing FBS, with addition of different concentrations of LPA or vehicle only at the same time. FBS-induced differentiation was observed by immunocytochemical and Hoechst 33342 staining techniques on the 7th day after exposure to LPA. Figure 3 presented a set of representative microphotographs taken from one of the vehicle control group, showing how we could calculate the percentage changes of two kinds of newly-differentiated neuronal progenitors in the control and LPA-treatment groups. MAP2-positive neurons (red-stained in Fig.3A) were distinguished from ChAT-positive neurons (green-stained in Fig.3B), with clearly identifiable cell body contours and dendrites in both cases; and thus the percentages of these two kinds of neurons could be calculated by the ratio of their numbers to the total number of cells in the culture (as revealed by the Hoechst 33342-stained blue nuclei)(Fig.3D), possibly including the nuclei of undifferentiated NSCs, newly-generated neuronal and glial cells, and so on. Figure 3 also showed that all ChAT-positive neurons should be MAP2-positive, while we could not find even a single ChAT-labeled neuron following treatment of two related antisera, implying that all cholinergic-committed neuronal progenitors should be derived from more primitive and unspecific MAP2-positive ones.
Histograms in Fig.4 showed the results of calculations obtained from seven LPA-treatment groups (0.05~3.0 μmol/L of LPA) and one control group (vehicle only). It was observed that, as compared to that in the vehicle control groups, application of 0.05 μmol/L LPA elicited a detectable percentage increases in both MAP2- and ChAT-positive neurons. The promotive effects increased as the dosage of LPA gradually increased, with a peak at 0.1 μmol/L LPA (40% for MAP2-positive and 25% for ChAT-positive neurons) (P<0.01 in two cases) as compared to the corresponding 26% and 7% in the vehicle groups; moreover, as LPA was higher than 0.1 μmol/L, the promotive effects on both neurons declined, and at last 2.0~3.0 μmol/L LPA even elicited decreases in their percentages as compared to that in the control groups. Figure 4 also showed that the percentages of MAP2-positive neurons in all LPA-treatment and control groups were always higher than that of ChAT-positive ones. However, it seemed that the increasing rate of ChAT-positive neurons were always faster than that of MAP2-positive ones when the concentration of LPA was successively increased towards 0.1 μmol/L; and at the same time, the percentage of ChAT-positive neurons could not surpass one third of the total cultured cells even the optimum concentration of LPA was used, at which the MAP2-positive neurons reached the peak, not more than 40% of the total cells.
2.2.2 Detection of MAP2 and ChAT proteins by Western blot
As a complementary support to the results presented in Fig.4, Western blot of two relevant marker proteins, MAP2 and ChAT, also showed similar changes following LPA treatment. In these cases we could get the quasi-quantitative changes of marker proteins rather than the percentage changes in number of two different neurons, excluding the possibilities that an increased percentage might be concomitant with a decreased real number of these neurons or vice versa. As shown in Fig.5A, the expression of MAP2 appeared as early as 24 h after LPA treatment, then increased in the following days and peaked on the 7th day. At the same time, when 1.0 mmol/L LPA was added, a time-dependent but less expressed MAP2 protein on all four examining days was observed as compared to that in 0.1 mmol/L LPA group. On the other hand, ChAT expression exhibited a quite different time course in two LPA-treatment and even in the control groups, in which ChAT expression could only be detected late on the 5th day, and then increased on the 7th day following LPA or vehicle application. Thus, the data presented in Fig.4 and Fig.5 achieved similar suggestions that 0.1 mmol/L might be the optimum concentration of LPA for promoting the generation of both MAP2- and ChAT-positive neurons, though the latter ones markedly fell behind the former ones, indicating the possibility that the expression of ChAT could appear only on the basis of MAP2 expression.
2.3 Effects of LPA on migration and neurite extension of cells derived from NSCs
Some studies supported the proposal that LPA promoted the differentiation of neuronal progenitors from NSCs by observing the effects of LPA on general morphological changes of neuron-committed as well as glia-committed (reported elsewhere) progenitors by phase contrast microscopy. We could not distinguish two of them in these observations due to the limit of technique. Microphotographs in Fig.6 showed that when the cultures were treated with FBS and vehicle only, there appeared a few smaller neurospheres with two or three progenitor cells migrating out of them as observed 24 h later (Fig.6A); while as 1.0 μmol/L LPA was added at the same time point, more cells migrated from a larger neurosphere with the shape changing from round to spindle (Fig.6B); moreover, after 48 h exposure to 1.0 μmol/L LPA, there was a robust migration of cells from neurospheres with smaller size and longer neurites in general (Fig.6D). These morphological features showed that LPA at certain lower concentrations might also promote the migration and neurite extension in the early-differentiated neural progenitors.
3 DISCUSSION
The present study for the first time provided evidence that LPA exhibited a dual or bi-directional action on the proliferation of rat embryonic NSCs in vitro according to the dosages used in the cultural medium. These results were in consistent with that obtained in our previous studies on the effects of LPA upon the apoptosis induced by different environmental insults in cultured mature cortical neurons[32,33]. It is suggested that a lower dosage of LPA acts as a survival factor in the apoptotic processes while a higher dosage itself induces neuronal apoptosis. We do not know exactly why the higher and lower dosage LPA has opposite effect on the survival of NSCs. Since lower LPA also promoted the differentiation of NSCs, more complicated and sophisticated processes should be involved in the action of lower LPA. Thus, we inclined to suggest that the suppressive effect of higher LPA on the proliferation (and even the differentiation) of NSCs may be induced simply by its apoptogenic action as exhibited in the case of mature cortical neurons[32]. These problems need to be clarified further.
The present study further demonstrated that LPA within certain lower concentration range promoted the differentiation of MAP2- and ChAT-positive neurons from embryonic NSCs, and 0.1 μmol/L seems to be the optimum concentration of LPA for harvesting the most unspecific MAP2-positive and specific cholinergic neurons among the total cells in the medium. At the same time, Western blot assays provided complementary support that LPA actually increased the real number of two different neurons, not only the percentages of them. We also found some important and interesting differences in the expression of ChAT as compared to that of MAP2. MAP2 appeared as early as 24 h after the addition of FBS with LPA in two LPA-treatment groups and even in vehicle control group, and 0.1 μmol/L was the optimum dosage of LPA for exhibiting a daily-intensified MAP2 expression in the following days; in the case of ChAT expression, although LPA also intensified the ChAT expression related to the dosage and exposure time to LPA, this marker protein could only be detected late but abruptly on the 5th day after LPA application in two experimental and control groups, with the highest expression in the 0.1 mmol/L LPA group. These facts showed that addition of LPA might enhance the ChAT expression quantitatively but could not make this process happen earlier. Even the optimum concentration of LPA could not shorten the time-lag of the intensified ChAT expression temporally as compared to that in the vehicle control group. At the present time, it is difficult to explain why LPA affects the expressions of MAP2 and ChAT with different time courses. A possible explanation might be that there exists a complex and time-exhausting cascade of regulation for a series of genomic activations that are leading to the ChAT expression at the last stage, while these processes could not be enhanced by LPA at their earlier steps. Nevertheless, interestingly we have noticed the report presented recently by Ito et al.[11], indicating that neurotrophins could induce ChAT and other neurotransmitter-synthesizing enzymes even in the undifferentiated proliferating NSCs. Evidently, different bioactive agents might act at different steps that at last are leading to the appearance of functionally-specific neurons, such as cholinergic neurons. It is also difficult to explain why the differentiation of NSCs is more sensitive than their proliferation to LPA application and why the expression of ChAT on the 5th day is weaker in 0.1 μmol/L LPA group than that in the control group. Probably the underlying mechanisms are subtle and complicated. These issues need to be evaluated further under different experimental conditions. Based on the experiments carried out mainly on fibroblasts, Moolenaar[34] and van Leeuwen et al.[14] have analysed the possible signaling pathways by which LPA exhibits promotive action at the cellular level. It is indicated that at least three subtypes of LPA receptors and four G-protein-coupled signaling pathways are involved in the mitogenic action of LPA. In the nervous system, Contos et al.[18] also emphasized the involvement of LPA1 and LPA2 receptors in the neurogenesis; at the same time, Kingsbury et al.[35] reported recently that intact mouse cerebral cortices exposed to extracellular LPA ex vivo rapidly increased in width and production, and they argued that the growth was not due to increased proliferation but to the reduced receptor-dependent cell death and the increased terminal mitosis of neural progenitor cells. The mechanism(s) underlying the effects of LPA on the proliferation of NSCs and the neurogenesis in general also await more detailed elucidation.
In conclusion, LPA within certain lower range of concentrations may act as an extracellular signal promoting the proliferation of NSCs and their differentiation to unspecific MAP2-positive and specific cholinergic-committed neurons. Therefore, this small phospholipid might be helpful for establishing in vitro resources of NSCs and some neuronal and glial progenitor cells, including that differentiating specifically to cholinergic neurons.
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